The thesis deals with a utilization of hydrogels made of humic acids in both basic and applied research of this valuable natural material. The attention is paid to an interaction between the humic gel and cupric ions as the model heavy metal. The main experimental part focuses on an optimation of simple laboratory diffusion methods which serve as an innovative tool for modeling pollutants’ transport in natural humic environments. Various techniques were used in order to determine a diffusion coefficient of cupric ions in humic gel; the value is closely linked with the studied interaction between solid content of the gel and the diffusing species. Consequently, the diffusivity can be used as a standard parameter for basic reactivity mapping studies concerning humic substances. The final chapter of the thesis deals with a preparation of mixed reversible hydrogels formed by a reaction between humic acids and chitosan. These materials represent a suitable colloidal form for humic acids’ industrial and agricultural applications.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19010号 / 工博第4052号 / 新制||工||1623(附属図書館) / 31961 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 田畑 泰彦, 教授 岩田 博夫, 教授 木村 俊作 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19746号 / 工博第4201号 / 新制||工||1648(附属図書館) / 32782 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 田畑 泰彦, 教授 秋吉 一成, 教授 木村 俊作 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

No description available.

Hyaluronic acid is an organic polysaccharide with a wide range of uses in medical and cosmetic industries due to its physiological properties. Crosslinked hyaluronic acid is a commonly used filler agent because of its water retention capabilities. N-deacetylation can be performed to enable new derivatives of hyaluronic acid. Both chemical and enzymatical approaches were investigated in this literature study to find methods retaining a high molecular weight product. Chemical N-deacetylation of hyaluronic acid has significant challenges with being treated by acid or base while both preventing degradation and maintaining its molecular weight. The method that seems the most promising is treating hyaluronic acid with hydroxylamine. Another method is enzymatic N-deacetylation. It was found that an enzyme N-deacetylated hyaluronic acid in female breast skin from 69-year-olds and above. The isolated enzyme had molecular weights ranging from 63 kDa to 79 kDa. Another enzyme that was produced recombinantly proved to be efficient since it retained high molecular weight and had a degree of deacetylation of 10.1 %. Today there exists only a few methods for crosslinking deacetylated hyaluronic acid. However, for chitosan, there are multiple methods available for crosslinking. Since it uses similar reactions that could be applicable to that of deacetylated hyaluronic acid. Reacetylation of the free amino groups has proven to be possible after crosslinking with a simple and cheap method resulting in an almost complete reacetylation. NMR proved to be an adequate method for analyzing the degree of deacetylation and higher-order structures. HPLC-UV spectroscopy may be used to increase the credibility of the analysis.

Hydrogels are three-dimension networks of water-soluble polymer chains and have attracted interest in biomedical engineering, targeted drug delivery, tissue engineering, and regenerative medicine due to their ability to retain water coupled with their highly tunable physicochemical and biological properties. In the specific context of wound care, hydrogels can both maintain high wound hydration as well as absorb and manage wound exudate, both of which are major challenges in wound care. Hydrogel wound dressings can simultaneously deliver medication directly to the wound to suppress or treat infections, including antibiotic-resistant strains such as Methicillin-resistant S. aureus (MRSA). This thesis develops two wound care products that can address challenges in the selection and delivery of drugs to treat antibiotic-resistant strain infections: (1) in situ-gelling poly(oligoethylene glycol methacrylate) (POEGMA) hydrogel wound dressings containing self-assembled nanoparticles encapsulated with fusidic acid; and (2) an in situ calcium-crosslinked alginate scaffold produced using pressurized gas expanded liquids (PGX) technology impregnated with fusidic acid or tigecycline using supercritical adsorptive precipitation (sc-AP). The POEGMA hydrogel wound dressings helped supress MRSA infection and prevent systemic infection during the course of treatment, facilitating a 1-2 fold decrease in bacterial load in the wound bed. The sc-AP technology was shown to be compatible with loading clinically-relevant doses of both antimicrobial compounds, while the resulting wound dressings were effective in treating MRSA wound infections. In case of tigecycline loaded alginate scaffolds, the infection was completely cleared. In tissue engineering applications, injectable macroporous hydrogels are particularly limited by two factors: (1) their need for invasive administration, typically implantation; and (2) their generally weak mechanics. In the first case, reports of injectable hydrogels often involve toxic compounds or by-products that result in loss of cell viability. This thesis addresses this challenge by design and development of a POEGMA-based macroporous hydrogel scaffold based on a novel, non-cytotoxic pore forming emulsion based on perfluorocarbons. Use of the pore-forming emulsion significantly improved cell viability in vitro 14 days after injection and was well tolerated in vivo with minimal to no inflammatory response. In the second case, an interpenetrating “hard-soft” nanofibrous hydrogel network was fabricated by co-electrospinning POEGMA with poly(caprolactone) (PCL). The PCL phase significantly enhanced the mechanical properties of the electrospun POEGMA hydrogel scaffold making handling and manipulating the scaffolds possible, while the presence of the POEGMA phase significantly improved the biological properties of PCL scaffolds in terms of supporting significantly enhanced cell proliferation and delayed bacterial adhesion. Collectively, the advances made in this work address key challenges in the application of hydrogels in tissue engineering and wound care, with future potential to be applied to solve practical clinical challenges. / Dissertation / Doctor of Philosophy (PhD) / Hydrogels have been studied in various applications like targeted drug delivery, tissue engineering, regenerative medicine, and medical devices due to their tunable nature and their capacity to retain water. In many of these applications the pore size and porosity are the key to the performance of a hydrogel in a given application. In particular, the rate at which nutrients or wastes can move through a hydrogel, the stiffness of a hydrogel, and the interactions of a hydrogel with cells are all strongly dependent on the porosity of a hydrogel. Therefore, many techniques have been developed to produce hydrogels with well-defined pore sizes, in particular “macroporous” hydrogels that have larger pores at or above the size of a cell. However, the typical techniques used to make such hydrogels often require additives or manufacturing steps that make them challenging to implement in different applications. This thesis addresses challenges in the fabrication of controllable porosity of hydrogels for applications in wound care (including the treatment of antibiotic-resistant infected wounds) and regenerative medicine, in the latter case enabling minimally invasive injection of a macroporous hydrogel as well as enhancing its mechanics to better mimic native tissues. Each of these solutions aims to bring effective novel treatments to patients, offering alternative therapies for existing challenges in healthcare.

No description available.

<p>  </p> <p>Pluripotent stem cells (PSCs) are increasingly utilized to investigate early human developmental processes including gastrulation and organogenesis of endoderm-derived pancreatic lineages. Critical for tissue development, the PSC niche is a dynamic environment consisting of extracellular matrix (ECM) components that guide cell proliferation, migration, and differentiation. However, investigation of the interplay between the PSC niche and organogenesis has been limited to conventional two-dimensional (2D) cell culture or three-dimensional (3D) platforms requiring use of ill-defined materials (e.g., Matrigel). Furthermore, these systems lack tunability to probe specific qualities of the PSC niche including mechanical properties and biochemical compositions. In this dissertation, modular and dynamic hydrogels were designed to study PSC and niche interactions during differentiation and pancreatic organogenesis. Specifically, two bioorthogonal chemical reactions, thiol-norbornene photopolymerization and tetrazine-norbornene inverse electron demand Diels-Alder (iEDDA) reactions were employed to generate gelatin- and poly(ethylene glycol) (PEG)-based hydrogels with spatiotemporally tunable physicochemical properties. Following mechanical characterization of the hydrogels, the multicomponent gelatin-based hydrogels were assessed for supporting viability and pluripotency of human induced pluripotent stem cells (hiPSCs), as well as for permitting their trilineage differentiation. Next, fully synthetic PEG-based hydrogels with temporally tunable crosslinking density were established to probe the effect of matrix mechanics on definitive endoderm differentiation of the hiPSCs. Finally, hiPSC-to-pancreatic progenitor cell differentiation was explored in both naturally-derived gelatin-based hydrogels and synthetic PEG-based hydrogels, with cells differentiated on a 2D surface as a control. Overall, this work demonstrates that culture dimensionality, material compositions, and mechanics profoundly influence hiPSC differentiation and pancreatic morphogenesis.</p>

Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer with an average survival time of 15 months. GBM is considered incurable with even the most aggressive multimodal therapies and is characterized by near universal recurrence. Irreversible electroporation (IRE) is a cellular ablation method currently being investigated as a therapy for a variety of cancers. Application of IRE involves insertion of electrodes into tissue to deliver pulsed electric fields (PEFs), which destabilize the cell membrane past the point of recovery, thereby inducing cell death. While this treatment modality has numerous advantages, the lack of selectivity for malignant cells limits its application in the brain where damage to healthy tissue is especially deleterious. In this dissertation we hypothesize that a form of IRE therapy, high-frequency IRE (H-FIRE), may be able to act as a selective targeted therapy for GBM due to its ability to create an electric field inside a cell to interact with altered inner organelles. Through a comprehensive investigation involving experimental testing combined with numerical modeling, we have attained results in strong support of this hypothesis. Using tissue engineered hydrogels as our platform for therapy testing, we demonstrate selective ablation of GBM cells. We develop mathematical models that predict the majority of the electric field produced by H-FIRE pulses reach the inside of the cell. We demonstrate that the increased nuclear to cytoplasm ratio (NCR) of malignant GBM cells compared to healthy brain—evidenced in vivo and in in vitro tissue mimics—is correlated with greater ablation volumes and thus lower electric field thresholds for cell death when treated with H-FIRE. We enhance the selectivity achieved with H-FIRE using a molecularly targeted drug that induces an increase in NCR. We tune the treatment pulse parameters to increase selective malignant cell killing. Finally, we demonstrate the ability of H-FIRE to ablate therapy-resistant GBM cells which are a focus of many next-generation GBM therapies. We believe the evidence presented in this dissertation represents the beginning stages in the development of H-FIRE as a selective therapy to be used for treatment of human brain cancer. / Ph. D.

Understanding cell migration is a key step in unraveling many physiological phenomena and predicting several pathologies, such as cancer metastasis. In particular, confinement has been proven to be a key factor in the cellular migration strategy choice. As our insight in the field improves, new tools are needed in order to empower biologists’ analysis capabilities. In this framework, microfluidic devices have been used to engineer the mechanical and spatial stimuli and to investigate cellular migration response in a more controlled way. In this work, we will review the existing technologies employed in the realization of microfluidic cellular migration assays, namely the soft lithography of PDMS and hydrogels and femtosecond laser micromachining. We will give an overview of the state of the art of these devices, focusing on the different geometrical configurations that have been exploited to study specific aspects of cellular migration. Our scope is to highlight the advantages and possibilities given by each approach and to envisage the future developments in in vitro migration studies under spatial confinement in microfluidic devices.